【摘要】 采用基因定点突变的方法,构建了细菌视紫红质(Bacteriorhodopsin,BR)的3种突变体蛋白,即单突变体BRE194Q、三突变体BRI119T/T121S/A126T和四突变体BRI119T/T121S/A126T/E194Q.测定了突变体和野生型BR在水溶液和聚乙烯醇(PVA)膜中的紫外-可见吸收光谱和拉曼光谱,采用显微视频录像技术记录了PVA膜中野生型和3个突变体样品的M态寿命.与野生型BR相比较,在水溶液中,单突变体的可见吸收光谱的最大吸收峰发生了轻微红移,三突变体和四突变体的最大吸收峰则分别发生了11.0和12.0 nm的明显蓝移.在PVA膜中,3个突变体BR的可见吸收光谱的最大吸收峰均发生蓝移,四突变体BR的最大吸收峰为557 nm,蓝移达15.0 nm.四突变体BR在水溶液中的共振拉曼光谱不仅表现有与M态特征相关的1567和1573 cm-1谱带,还有L态特征带1334 cm-1及N态特征带1200,1328,1530和1549 cm-1.在PVA膜中的样品与在水溶液中的比较,四突变体共振拉曼光谱的1334和1549 cm-1带消失,同时1187 cm-1带的强度下降.显微视频录像技术记录的PVA膜中样品的M态寿命表明,野生型BR的M态寿命最短,单突变体的M态寿命小于1.0 s,三突变体的寿命为3.0 s,四突变体的寿命为2.0 s.更多还原

【Abstract】 The site-specific residue replacements E194Q,I119T/T121S/A126T and I119T/T121S /A126T/E194Q were introduced into bop gene and expressed in halobacterium salinarium L33 with the vector pXL-NovR,respectively.The photoreaction of three mutants BRE194Q,BRI119T/T121S/A126T and BRI119T/T121S/A126T/E194Q(the quadruple mutant) of bacteriorhodopsin(BR) and WT-BR(wild type BR) was investigated in solution and in PVA films at pH=7.0 by UV-Vis absorption spectrum and Resonance Raman spectra.The lifetime of M intermediate of the three mutants in PVA films were recorded by employing photomicrography.In distilled water,the visible absorption maximum of BRI119T/T121S/A126T and BRI119T/T121S/A126T/E194Q mutants were blue shifted notably by 11 and 12 nm respectively and the BRE194Q ′s was weakly red shifted in comparison with WT-BR.In PVA film,BRI119T/T121S/A126T/E194Q mutant caused a blue shift of the visible absorption spectrum from 568 to 557 nm.The characteristic bands of Resonance Raman spectra of BRI119T/T121S/A126T/E194Q mutant were observed at 1334cm-1 of the L intermediate,1200,1328,1530 and 1549 cm-1 of the N intermediate and 1567 and 1573 cm-1 of M intermediate in solution.However,the 1334 and 1549 cm-1 bands of the quadruple mutant disappeared and the intensity of 1187 cm-1 band decreased in PVA film.The lifetime of M intermediate of BRE194Q was less than 1.0 s by photomicrography.The lifetime of M intermediates of BRI119T/T121S/A126T and BRI119T/T121S/A126T/E194Q were 3.0 s and 2.0 s,respectively.更多还原